Nuclear resonance vibrational spectroscopy (NRVS) and density functional theory calculation (DFT) have been applied to illuminate the effect of axial ligation on the vibrational dynamics of iron in heme carbonyl. The analyses of the NRVS data of five- (5c) and six-coordinate (6c) heme-CO complexes indicate that the prominent feature of 57Fe partial vibrational density of state (57FePVDOS) at the 250-300 cm-1 region is significantly affected by the association of the axial ligand. The DFT calculations predict that the prominent 57FePVDOS is composed of iron in-plane motions which are coupled with porphyrin pyrrole in-plane (ν49, ν50, and ν53), an out-of-plane (γ8) (two of four pyrrole rings include the in-plane modes, while the rest of pyrrole rings vibrate along the out-of-plane coordinate), and out-of-phase carbonyl C and O atom displacement perpendicular to the Fe-C-O axis. Thus, in the case of the 5c CO-heme the prominent 57FePVDOS shows sharp and intense feature because of the degeneracy of the e symmetry mode within the framework of C4v symmetry molecule, whereas the association of the axial imidazole ligand in the 6c complex with the lowered symmetry results in split of the degenerate vibrational energy as indicated by broader and lower intensity features of the corresponding NRVS peak compared to the 5c structure. The vibrational energy of the iron in-plane motion in the 6c complex is higher than that in 5c, implying that the iron in the 6c complex includes stronger in-plane interaction with the porphyrin compared to 5c. The iron in-plane mode above 500 cm-1, which is predominantly coupled with the out-of-phase carbonyl C and O atom motion perpendicular to Fe-C-O, called as Fe-C-O bending mode (δFe-C-O), also suggests that the 6c structure involves a larger force constant for the e symmetry mode than 5c. The DFT calculations along with the NRVS data suggest that the stiffened iron in-plane motion in the 6c complex can be ascribed to diminished pseudo-Jahn-Teller instability along the e symmetry displacement due to an increased a1-e orbital energy gap caused by σ* interaction between the iron dz2 orbital and the nitrogen p orbital from the axial imidazole ligand. Thus, the present study implicates a fundamental molecular mechanism of axial ligation of heme in association with a diatomic gas molecule, which is a key primary step toward versatile biological functions.